1. Field of the Invention
This invention relates to liquid chromatography and, more particularly, to chromatographic columns which have one or more ports that enable packing of the sorbent material into the column's chamber.
2. Description of the Related Art
A chromatographic column has an inner chamber that contains a particulate sorbent surrounded by sidewalls and two screens. The screens prevent the sorbent from escaping the inner chamber yet allow the free flow of liquid sample therethrough. The screens typically are made of mesh or porous membrane, commonly referred to as a frit.
Chromatographic separation of the components from a liquid sample occurs on the sorbent. Fine and reproducible separation of a liquid sample is accomplished through even flow distribution across a uniformly packed sorbent. Dead spaces and voids in the column or sorbent causes uneven flow of the liquid sample through the sorbent which produces so-called "chromatographic peak tailing".
Besides its chromatographic performance, the quality of a column is also determined by its convenience in packing, unpacking, cleaning, consistency in performance from one packing to another, and convenience in handling and storage.
Historically, two types of columns are used: fixed volume, and adjustable volume.
Fixed volume columns usually comprise a cylindrical tube filled with a sorbent with two end plates disposed over the opened ends of the cylindrical tube. Fixed volume columns are useful because they provide consistent chromatographic conditions and allow for easy data comparison from different experiments and between different laboratories. Fixed volume columns are also less expensive than adjustable volume columns.
One major disadvantage of fixed volume columns is their cumbersome packing procedure which requires the use of an extension chamber installed over one end of the cylindrical tube in place of an end plate. After the cylindrical tube has been packed with sorbent, the extension chamber is then carefully removed and replaced with a frit and an end plate. Unfortunately, dead spaces or voids may be created in the column or sorbent when the extension chamber is replaced.
The problems associated with packing a fixed volume column have been addressed in U.S. Pat. Nos. 4,497,711 and 4,578,193 which disclose using a special valve that passes through an end plate and a porous bed-retaining membrane. Another method for packing the column is to create a sorbent slurry which is then feed through a narrow, filler tube attached to the sidewall of the column. Once the column is filled, additional sorbent is deposited inside the filler tube to form a seal that prevents the sorbent from escaping from the column.
Unfortunately, there are several disadvantages with columns that have filler tubes for packing sorbent. First, such columns are not reusable since the sorbent can not be removed from the column through the filler tube. Second, the sorbent may settle in the column over time thereby creating voids. Ideally, it would be desirable to pack additional sorbent into the column to fill these voids and to restore the column's performance. However, since the filler tube is sealed, packing additional sorbent into the column is precluded. Third, the filler tube creates a long and narrow dead space where a portion of the liquid sample can reside which can Cause peak tailing in the chromatographic separation. The column is hard to validate for sterile applications since, when this occurs, microorganisms can survive cleaning agents in the stale solution of the long and narrow filler tube.
The second type of chromatographic column, called adjustable volume column, includes a plunger which is adjusted in height and positioned against the sorbent to tightly pack the sorbent inside the column. The main advantage of adjustable volume columns over fixed volume columns is that the sorbent may be quickly and neatly packed inside the column. One drawback with these columns, however, is that the volume of the sorbent is difficult to reproduce. Another drawback is that the plungers themselves can create sealing problems, especially when large diameter columns are used.
In addition to the two general types of chromatographic columns mentioned above, chromatographic columns can also be classified according to the direction of liquid sample flow in the column--horizontal flow and vertical flow. The packing process is relatively easy and reliable in horizontal flow column designs as disclosed in U.S. Pat. No. 4,676,898 (Saxena). In the column design disclosed therein, the end plate is equipped with two packing ports for packing. Unlike the vertical flow columns, the liquid sample in horizontal flow columns flows perpendicular to the column's longitudinal axis. The sorbent is contained in the column by top and bottom flat end plates and on the sides by two circular frits coaxially positioned with respect to each other.
Besides the packing and unpacking convenience, the column design should allow for the easy transfer of chromatographic conditions from small to large size columns, known as "scaling up". When "scaling up", it is desirable to keep the thickness of the sorbent constant so that the volume of the sorbent is increased only by increasing the cross-sectional area of the column. When this is done, the back pressure and sample elution times will remain constant throughout the "scaling up" process. The solvent and sample volumes are then directly proportional to the column size.
Vertical flow columns which have relatively high ratios of cross-section to height dimensions are known as "pancake shaped" columns. In these columns, the end plates or plungers have to be thicker than the column's sidewalls to withstand the pressure which builds up inside the column. This makes large "pancake shaped" columns heavy and expensive to construct and difficult to service.
The flow distribution of a liquid sample in a large "pancake shape" column also presents problems. As shown in FIGS. 1A-1C, when a liquid sample enters the column through an inlet port located in the center of the column, it spreads evenly over the frit. The portion of the liquid sample that flows through the center of the sorbent follows a shorter path and reaches the outlet port before the portion of the sample mixture that travels along the sidewall, through the sorbent, and then back to the center to the outlet port. As a result, the flow of the liquid sample through the column is uneven which causes tailing of the chromatographic peaks, as illustrated in FIG. 2. To correct this tailing problem, special flow adapters must be used in the column.
In an attempt to improve flow distribution of the liquid sample through the column, plumbing ports may be disposed horizontally in the column as suggested in U.S. Pat. No. 4,722,786. This design includes a bell-shaped end plate placed over the frit. No prototype is available on the market for this design for testing on either a small or large scale basis. It is anticipated that an extremely large dead space is created under the end plate which will ruin separation due to mixing of the sample components.
Another important feature of a chromatographic column is the convenience of handling and storage. Most of the columns currently available, have liquid sample inlet and outlet ports positioned vertically in the center of the column. The location of these ports on the column necessitates suspending the column from a stand to prevent pinching or crushing of the lower plumbing tube under the weight of the column. If the column could support its weight by standing on its end plate, the need for a heavy stand could be eliminated. However, the vertical disposition of the plumbing ports located in the center of the flat end plate makes self-supporting columns impossible.
In summary, all chromatographic columns in the prior art have certain undesirable features that distinguish them from the chromatographic column disclosed herein. These undesirable features include:
(1) all must be packed and unpacked either from the top or the bottom opening but not through their sidewalls except for those which use filler tubes; PA0 (2) those having filler tubes are not reusable; PA0 (3) the inlet and outlet ports are disposed vertically on the bottom end plate which prevents them from being used directly on a horizontal surface; PA0 (4) all columns must use flow distributors which help distribute the liquid sample from a central located inlet port over the entire surface of the frit simultaneously to reduce peak tailing; and, PA0 (5) all columns require that the flow direction through the column be reversed to remove an air bubble from the top frit which can ruin the chromatographic separation, especially if the gradient elution was in progress.
Chromatographic columns which do not have these undesirable features would be highly desirable.